CN115427578A

CN115427578A - Gene therapy

Info

Publication number: CN115427578A
Application number: CN202180020240.6A
Authority: CN
Inventors: D.施密特; V.库兹穆克; M.萨里姆; G.韦尔什
Original assignee: University of Bristol; Syncona Management LLP
Current assignee: University of Bristol; Syncona Management LLP
Priority date: 2020-03-12
Filing date: 2021-03-12
Publication date: 2022-12-02
Also published as: US20240197917A1; GB202003618D0; IL296255A; WO2021181118A1; BR112022018036A2; AU2021234169A1; EP4118216A1; CA3171232A1; MX2022011295A; JP2023517934A

Abstract

A viral vector, wherein said viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

Description

Gene therapy

Technical Field

The present invention relates to a viral vector comprising a COL4A3, COL4A4 or COL4A5 transgene and a kidney specific promoter, and the use of the viral vector in the treatment of Alport syndrome.

Background

Alport Syndrome (AS) is a genetic condition affecting about 1 of 5,000-10,000 of all individuals in the continental europe and the united states. AS is also known AS familial nephritis, hereditary nephritis, thin basement membrane disease and thin basement membrane nephropathy. This condition typically occurs in childhood and is associated with a range of phenotypes including progressive loss of renal function and possibly hearing loss and ocular abnormalities.

AS is caused by pathogenic mutations in COL4A3, COL4A4 and COL4A5 genes, which lead to abnormalities in the collagen IV α 345 network of the basement membrane. The condition can be transmitted in an X-linked, autosomal dominant, or autosomal recessive pattern, where X-linkage is common and autosomal recessive and autosomal dominant account for about 15% and 20% of cases, respectively.

In all men with X-linked form and all men and women with autosomal recessive form, without treatment, renal disease progresses from microhematuria (microhematuria) to proteinuria, progressive renal insufficiency and end stage renal disease.

AS can be diagnosed by genetic testing, and current treatments include Angiotensin Converting Enzyme (ACE) inhibitors or Angiotensin Receptor Blockers (ARBs) to delay the onset of end stage renal disease. However, there is currently no way to prevent end-stage renal failure, and kidney transplantation is the only option.

There are significant challenges to overcome in developing successful AS gene therapy. First, COL4A5, COL4A3, and COL4A4 proteins have 1685, 1670, and 1690 amino acids, respectively, which make their transport through adeno-associated virus (AAV) vectors challenging due to the limited cargo capacity of AAV. The second major challenge is to successfully deliver gene therapy to podocytes in the glomerulus, which produce collagen IV in the glomerular basement membrane.

The present invention aims to provide a novel gene therapy vector that can effectively deliver COL4A3, COL4A4 or COL4A5 transgenes to podocytes, thereby providing a therapy for treating Alport syndrome.

Disclosure of Invention

The present invention provides a viral vector, wherein the viral vector comprises a COL4A3, COL4A4 or COL4A5 transgene. The viral vector can be used to target podocytes within the glomerulus in order to treat Alport syndrome.

Without being bound by theory, the inventors believe that podocytes provide a highly tractable target for gene therapy approaches in renal disease, and by targeting COL4A3, COL4A4, or COL4A5 to podocytes, the collagen IV α 345 network of the glomerular basement membrane can be altered and at least partially normalized.

In one aspect, the invention provides a viral vector, wherein the viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

The COL4A3 transgene may encode a COL4A3 polypeptide or fragment thereof, said COL4A3 polypeptide comprising a sequence identical to SEQ ID NO:1, or a polypeptide sequence having at least 70% identity to SEQ ID NO:1, a polypeptide sequence having at least 70% identity; the COL4A4 transgene may encode a COL4A4 polypeptide or fragment thereof, the COL4A4 polypeptide comprising a sequence identical to SEQ ID NO:2, or a polypeptide sequence having at least 70% identity to SEQ ID NO:2 having at least 70% identity; and/or the COL4A5 transgene may encode a COL4A5 polypeptide or fragment thereof, said COL4A5 polypeptide comprising an amino acid sequence identical to SEQ ID NO:3, or a polypeptide sequence having at least 70% identity to SEQ ID NO:3, having at least 70% identity. In some embodiments, the COL4A3 transgene encodes a full-length COL4A3 polypeptide, and the COL4A4 transgene encodes a full-length COL4A4 polypeptide; and/or the COL4A5 transgene encodes a full-length COL4A5 polypeptide. Suitably, the COL4A3, COL4A4 or COL4A5 transgene is human and/or comprises a Hemagglutinin (HA) tag.

Preferably, the viral vector comprises a podocyte-specific promoter. Suitably, the podocyte-specific promoter is the minimal nephrin promoter NPHS1 or podocin promoter NPHS2. In some embodiments, the podocyte-specific promoter is the minimal nephrin promoter NPHS1.

The inventors have developed a minimal nephrin promoter that is shorter than known minimal nephrin promoters and surprisingly capable of driving transgene expression in podocytes. The promoter also surprisingly retains podocyte specificity. This minimal nephrin promoter can be used to minimize cargo size and aid in packaging of full-length COL4A3, COL4A4 or COL4A5. Thus, the minimal nephrin promoter NPHS1 may comprise the sequence as set forth in SEQ ID NO:10 or a nucleotide sequence corresponding to SEQ ID NO:10, or a variant consisting of a sequence as set forth in SEQ ID NO:10 or a nucleotide sequence corresponding to SEQ ID NO:10 variant compositions that are at least 70% identical.

Suitably, the viral vector is an adeno-associated virus (AAV). Suitably, the AAV vector is in the form of an AAV vector particle. In some embodiments, the AAV vector particle is a podocyte-specific AAV vector. In some embodiments, the AAV vector is AAV serotype 2/9, LK03, or 3B.

In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene is a minigene (mini-gene).

In some embodiments, the viral vector further comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the viral vector does not comprise a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

In some embodiments, the viral vector further comprises a Kozak sequence between the promoter and the COL4A3, COL4A4, or COL4A5 transgene.

Suitably, the viral vector additionally comprises a polyadenylation signal, such as the bovine growth hormone (bGH) polyadenylation signal or the early SV40 polyadenylation signal. In some embodiments, the polyadenylation signal is the early SV40 polyadenylation signal.

In one aspect, the invention provides viral vector gene therapy, wherein the viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

In a preferred embodiment, the viral vector is a viral vector according to the invention.

In one aspect, the invention provides viral vector gene therapy, wherein the gene therapy comprises:

a first viral vector comprising at least a portion of a COL4A3, COL4A4, or COL4A5 transgene; and

a second viral vector comprising at least a portion of a respective COL4A3, COL4A4, or COL4A5 transgene.

In a preferred embodiment, the first viral vector is a viral vector according to the invention and/or the second viral vector is a viral vector according to the invention.

In one aspect, the invention provides a viral vector or viral vector gene therapy according to the invention for use in the treatment or prevention of Alport syndrome.

Suitably, the viral vector or viral vector gene therapy is administered to a human patient. In some embodiments, the viral vector or viral vector gene therapy is administered systemically. In some embodiments, the viral vector or viral vector gene therapy is administered by intravenous injection. In some embodiments, the viral vector or viral vector gene therapy is administered by injection into the renal artery.

Viral vectors

Adeno-associated virus (AAV) vectors

The viral vector may be an adeno-associated virus (AAV) and suitable AAV vector serotypes include 2/9, LK03 and 3B.

The viral vector may be in the form of an AAV vector particle.

AAV vector particles can be encapsulated by capsid proteins. The serotype may facilitate transduction of podocytes, e.g., specific transduction of podocytes. Preferably, the AAV vector particle is a podocyte-specific vector particle. AAV vector particles can be encapsulated by podocyte-specific capsids. The AAV vector particles can comprise podocyte-specific capsid proteins. Targeted transduction of podocytes should remove the effects of hepatic tropism (tropism) after systemic application.

Suitably, the AAV vector particle may be in the form of a trans-capsid (transduced) in which AAV genomes or derivatives having ITRs of one serotype are packaged in capsids of a different serotype. AAV vector particles also include chimeric (mosaic) forms in which a mixture of unmodified capsid proteins from two or more different serotypes make up the viral capsid. AAV vector particles also include chemically modified forms that carry ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

When the derivative comprises a capsid protein, i.e., VP1, VP2, and/or VP3, the derivative can be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the invention encompasses providing the capsid protein sequences of AAV from different serotypes, clades, clones or isolates (isolates) within the same vector (i.e. a pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle.

The chimeric, shuffled or capsid-modified derivatives are typically selected to provide one or more desired functions to the AAV vector. Thus, these derivatives may exhibit increased gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism ranges, and/or improved podocyte targeting compared to AAV vectors comprising a naturally occurring AAV genome. Increased gene delivery efficiency can be achieved by improved receptor or co-receptor binding on the cell surface, improved internalization, improved transport into the cell and into the nucleus, improved uncoating of the virion, and improved conversion of the single-stranded genome into a double-stranded form. Increased efficiency may also be associated with altered tropism ranges or targeting of podocytes, so that the carrier dose is not diluted for administration to tissues in which it is not needed.

Chimeric capsid proteins include those generated by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue method, in which a non-infectious capsid sequence of one serotype is co-transfected with capsid sequences of a different serotype, and targeted selection is used to select for capsid sequences with the desired properties. Capsid sequences of different serotypes can be altered by homologous recombination within the cell, thereby generating novel chimeric capsid proteins.

Chimeric capsid proteins also include those generated by engineering capsid protein sequences to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more different serotypes of capsid protein.

The shuffled or chimeric capsid protein can also be generated by DNA shuffling or by error-prone PCR. The hybrid AAV capsid gene can be created by: sequences of related AAV genes, such as sequences encoding capsid proteins of multiple different serotypes, are randomly fragmented and then reassembled in a self-priming polymerase reaction, which may also result in crossovers in regions of sequence homology. A library of hybrid AAV genes created by shuffling the capsid genes of several serotypes can be screened to identify viral clones with the desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to create a diverse library of variants, which can then be selected for desired properties.

The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions or insertions relative to the native wild-type sequence. In particular, the capsid gene may be modified by inserting sequences of unrelated proteins or peptides in the open reading frame of the capsid coding sequence or at the N and/or C ends of the capsid coding sequence. An unrelated protein or peptide may advantageously be one that acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or increasing the specificity of the vector to target a particular cell population. An unrelated protein may also be a protein that aids in the purification of the virus particle as part of the production process, i.e. an epitope or an affinity tag. Typically the insertion site is selected so as not to interfere with other functions of the viral particle, such as internalization, transport of the viral particle.

The capsid protein may be an artificial or mutated capsid protein. As used herein, the term "artificial capsid" refers to a capsid particle comprising an amino acid sequence that does not occur in nature or comprises an amino acid sequence that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein comprises a mutation or variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived, wherein the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned.

The capsid protein can comprise a mutation or modification relative to a wild-type capsid protein that increases the ability to transduce podocytes relative to unmodified or wild-type viral particles. The increased ability to transduce a podocyte can be measured, for example, by measuring expression of a transgene, e.g., GFP, carried by the AAV vector particle, wherein expression of the transgene in the podocyte correlates with the ability of the AAV vector particle to transduce the podocyte.

AAV9 serotype

AAV2/9 serotype showed significant tropism for neonatal and adult mouse kidneys, localized to glomeruli and tubules (Luo et al, 2011 picconi et al, 2014 schievenbusch et al, 2010), and AAV2/9 vector in combination with renal intravenous injection has been demonstrated to be suitable for kidney-targeted gene delivery (Rocca et al, 2014). AAV2/9 is thus a suitable vector for use in the viral vectors of the invention.

The AAV vector particles can comprise AAV9 capsid proteins. Suitably, the AAV vector particle may be encapsulated by an AAV9 capsid protein.

The AAV vector particles can comprise AAV9 VP1 capsid protein, AAV9 VP2 capsid protein, and/or AAV9 VP3 capsid protein. Suitably, the AAV vector particle may be encapsulated by an AAV9 VP1 capsid protein, an AAV9 VP2 capsid protein, and/or an AAV9 VP3 capsid protein. Suitably, the AAV vector particles may be encapsulated by AAV9 VP1, VP2, and VP3 capsid proteins.

Suitably, the AAV9 VP1 capsid protein may comprise an amino acid sequence as set forth in SEQ ID NO:31 or an amino acid sequence substantially identical to SEQ ID NO:31, or consists of, a variant that is at least 90% identical.

Exemplary AAV9 VP1 capsid protein (SEQ ID NO: 31):

suitably, the variant may be identical to SEQ ID NO:31 are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

Suitably, the AAV9 VP2 and VP3 capsid proteins may be SEQ ID NOs: 31, or an N-terminal truncation compared to SEQ ID NO:31 is at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical.

AAV LK03 serotype

Synthetic AAV capsids such as LK03 may also be suitable vectors for use in the viral vectors of the invention. This vector has been shown to efficiently transduce human primary hepatocytes in vitro and in vivo. However, it has not been used for kidney targeted gene delivery until now. Surprisingly, AAV-LK03 vectors can achieve nearly 100% high transduction in human podocytes in vitro and can be used to specifically transduce podocytes in vitro (see PCT/GB 2020/050097).

The AAV-LK03 cap sequence consists of fragments from seven different wild-type serotypes (

AAV

1, 2, 3B, 4, 6, 8, 9) and is described in LiSowski, L., et al, 2014.Nature,506 (7488), pp.382-386, although AAV-3B accounts for 97.7% of the cap gene sequence and 98.9% of the amino acid sequence.

AAV vector particles may comprise LK03 capsid protein. Suitably, the AAV vector particles may be encapsulated by LK03 capsid protein.

AAV vector particles can be made from a composition comprising LK03 VP1 capsid protein, LK03 VP2 capsid protein, and/or LK03 VP3 capsid protein. Suitably, the AAV vector particle may be encapsulated by LK03 VP1 capsid protein, LK03 VP2 capsid protein, and/or LK03 VP3 capsid protein. Suitably, AAV vector particles may be encapsulated by LK03 VP1, VP2, and VP3 capsid proteins.

Suitably, the LK03 VP1 capsid protein may comprise a sequence as set forth in SEQ ID NO:32 or an amino acid sequence substantially identical to SEQ ID NO:32, or a variant consisting of a sequence as set forth in SEQ ID NO:32 or an amino acid sequence substantially identical to SEQ ID NO:32 a variant composition that is at least 90% identical.

Exemplary LK03 VP1 capsid protein (SEQ ID NO: 32):

suitably, the variant may be identical to SEQ ID NO:32 are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

Suitably, the LK03 VP2 and VP3 capsid proteins may be SEQ ID NO:32, or an N-terminal truncation thereof to SEQ ID NO:32 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

AAV3B serotype

AAV-3B is also known for its human hepatocyte tropism and is another suitable vector for use in the viral vectors of the invention. To date, it has not been used for kidney targeted gene delivery.

The AAV vector particle may comprise an AAV3B capsid protein. Suitably, the AAV vector particle may be encapsulated by an AAV3B capsid protein.

Two different isolates of AAV3 (AAV 3A and AAV 3B) have been cloned. AAV3 vectors are thought to be unable to efficiently transduce most cell types compared to vectors based on other AAV serotypes. AAV3B, however, can efficiently transduce podocytes. AA3B has been described in Rutledge, E.A., et al, 1998.Journal of virology,72 (1), pp.309-319.

The AAV vector particles can comprise an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and/or an AAV3B VP3 capsid protein. Suitably, the AAV vector particle may be encapsulated by an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and/or an AAV3B VP3 capsid protein. Suitably, the AAV vector particles may be encapsulated by AAV3B VP1, VP2, and VP3 capsid proteins.

Suitably, the AAV3B VPl capsid protein may comprise the amino acid sequence as set forth in SEQ ID NO:33 or an amino acid sequence substantially identical to SEQ ID NO:33, or a variant consisting of a sequence as set forth in SEQ ID NO:33 or an amino acid sequence substantially identical to SEQ ID NO:33 a variant composition that is at least 90% identical.

Exemplary AAV3B VP1 capsid protein (SEQ ID NO: 33):

suitably, the variant may be identical to SEQ ID NO:33 are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

Suitably, the AAV3B VP2 and VP3 capsid proteins may be SEQ ID NOs: 33, or an N-terminal truncation compared to SEQ ID NO:33, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.

AAV genome

The AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof.

The AAV genome is a polynucleotide sequence that can encode functions required for production of AAV particles. These functions include functions that play a role in the replication and packaging cycle of AAV in the host cell, including the encapsulation of the AAV genome in AAV particles. Naturally occurring AAV is replication-defective and relies on providing helper functions in trans to complete the replication and packaging cycle. Thus, the AAV genome used in the present invention is typically replication-defective.

The AAV genome may be in single-stranded form, either positive or negative, or double-stranded form. The use of a double stranded form allows bypassing the DNA replication step in the target cell and thus can accelerate transgene expression. The maximum packing capacity of the single-stranded form is greater than that of the double-stranded form. Suitably, the AAV genome is in single stranded form.

AAV, which occurs in nature, can be classified according to various biological systems. The AAV genome can be from any naturally derived AAV serotype, isolate, or clade.

AAV may be referred to according to its serotype. Serotypes correspond to a variant subspecies of AAV, which has unique reactivity due to the expression profile of its capsid surface antigen, which can be used to distinguish it from other variant subspecies. In general, AAV vector particles with a particular AAV serotype do not cross-react efficiently with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV vector of the invention may be an AAV3B, LK03, AAV9, or AAV8 serotype.

AAV may also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of naturally derived AAVs and generally refers to the phylogenetic group of AAVs that can be traced back to a common ancestor, and includes all progeny thereof. Furthermore, AAV may be referred to in terms of a particular isolate, i.e., a genetic isolate of a particular AAV found in nature. The term genetic isolate describes a population of AAV that is subject to limited genetic mixing with other naturally occurring AAV, thereby defining at the genetic level a population that is identifiably distinct.

Typically, the AAV genome of a naturally derived serotype, isolate, or clade of AAV comprises at least one Inverted Terminal Repeat (ITR). The ITR sequences provide a functional origin of replication in cis and allow integration and excision of the vector from the cellular genome. The ITR may be the only sequence required in cis next to the therapeutic gene.

The AAV genome may also comprise packaging genes, such as rep and/or cap genes that encode AAV particle packaging functions. The promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, p5 and p19 promoters are commonly used to express the rep gene, while the p40 promoter is commonly used to express the cap gene. The Rep gene encodes one or more of the proteins Rep78, rep68, rep52 and Rep40, or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2 and VP3 or variants thereof. These proteins constitute the capsid of the AAV particle, which determines the AAV serotype. VP1, VP2 and VP3 can be produced by alternative mRNA splicing (Trempe, J.P.and Carter, B.J.,1988.Journal of virology,62 (9), pp.3356-3363). Thus, VP1, VP2 and VP3 may have the same sequence, but wherein VP2 is truncated at the N-terminus relative to VP1 and VP3 is truncated at the N-terminus relative to VP 2.

The AAV genome can be the entire genome of a naturally occurring AAV. For example, a vector comprising a complete AAV genome can be used to prepare an AAV vector or vector particle.

Preferably, the AAV genome is derivatized for administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known AAV genomic derivative, as well as derivatives that can be generated by applying techniques known in the art. The AAV genome can be any naturally occurring derivative of AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. Suitably, the AAV genome is a derivative of AAV 2.

Derivatives of the AAV genome include any truncated or modified form of the AAV genome that allows for expression of a transgene from an AAV vector of the invention in vivo. Typically, the AAV genome can be significantly truncated to include minimal viral sequences, but still retain the functions described above. This is preferred for safety reasons to reduce the risk of recombination of the vector with the wild-type virus and also to avoid triggering of a cellular immune response due to the presence of viral gene proteins in the target cells.

Thus, the following moieties may be removed in the derivatives of the invention: an Inverted Terminal Repeat (ITR) sequence, replication (rep) and capsid (cap) genes. However, the derivative may additionally include one or more rep and/or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19 and exhibits a negligible random integration frequency, and thus can tolerate the retention of integration capability in AAV vectors in a therapeutic setting.

The invention further encompasses sequences that provide the AAV genome in a different order and configuration than the native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences of another virus or a chimeric gene consisting of sequences from more than one virus. Such chimeric genes may be composed of sequences of two or more related viral proteins from different viral species.

Minigene (mini-gene) method

COL4A3, COL4A4, and COL4A5 have nearly 1700 amino acids, which are challenging to package in full-length form into AAV vectors due to AAV packaging limitations. However, the inventors have developed a minimum nephrin promoter that is shorter than known minimum nephrin promoters and surprisingly capable of driving transgene expression in podocytes. Such a minimal nephrin promoter can be used to minimize cargo size and aid in packaging of full-length COL4A3, COL4A4, or COL4A5.

An alternative to packaging full length COL4A3, COL4A4 or COL4A5 would be to provide the COL4A3, COL4A4 or COL4A5 transgene with a minigene. The minigene approach has been successfully used to develop gene therapy for the treatment of duchenne muscular dystrophy (Kodippili et al, 2018). In this approach, the transgene is truncated to fit the vector without losing the activity of the protein encoded by the transgene.

COL4A3, COL4A4 and COL4A5 proteins are homologous polypeptides of approximately 170-185kDa containing collagen Gly-X-Y repeats frequently spaced by noncollagen sequences and forming triple helix repeats. Each polypeptide also contains a large globular non-collagenous domain at the carboxy terminus. Approximately 200-300 amino acids should be removed from each of the COL4A3, COL4A4, and COL4A5 polypeptides to produce truncated transgenes suitable for use in the minigene approach. Amino acids may be removed from triple helix repeats. Preferably, no amino acids are removed from the non-collagenous region.

The COL4A5 minigene with an N-terminal HA tag or an N-terminal MyC tag can be ligated into AAV2/9, AAV LK03 and AAVL3 vectors containing the human minimal nephrin promoter (NPHS 2).

Viral vector gene therapy

The present invention provides viral vector gene therapy, wherein the viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

The viral vector used in viral vector gene therapy may be any of the viral vectors of the present invention described herein. Thus, it is to be understood that when reference is made herein to a viral vector, this may also refer to viral vector gene therapy, unless the context indicates otherwise.

Dual vector process

An alternative to viral vector gene therapy may be the use of a two-vector approach. In this method, the viral vector gene therapy comprises a first viral vector comprising at least a portion of a COL4A3, COL4A4, or COL4A5 transgene, and a second viral vector; and optionally a podocyte-specific promoter; the second viral vector comprises at least a portion of a respective COL4A3, COL4A4, or COL4A5 transgene; and optionally a podocyte-specific promoter. In other words, the transgene is divided into two separate sequences, each of which can be integrated into viral vector gene therapy as described herein. AAV two-vector methods are described, for example, in McClements and MacLaren 2017, incorporated herein by reference. The transgene sequences used in the two-vector approach may have overlapping exon or intron sequences that, when transduced, will combine by, for example, homologous recombination to reform a single transgene sequence. Alternatively, the two sequences may not overlap, but will be combined by, for example, intein protein trans-splicing methods. It is also possible to incorporate a splice donor signal in one of the two vectors and a splice acceptor signal in the second vector, which allows for trans-splicing following ITR-mediated head-to-tail concatemerization, resulting in mature mRNA. It is further possible to combine these methods into various hybrid methods, for example, which combine recombination with trans-splicing.

The first viral vector may be a viral vector according to the invention as described herein and/or the second viral vector may be a viral vector according to the invention as described herein. In a preferred embodiment, both the first and the second viral vector are viral vectors according to the invention as described herein.

COL4A3, COL4A4 and COL4A5 transgenes

The COL4A3, COL4A4, or COL4A5 transgene may comprise introns or intron sequences, which may be used to improve gene expression. Introns or intron sequences can be used in minigene or dual vector approaches. In the two-vector approach, this may allow recombination of the first and second portions of the transgene by homologous sequences of the intron. This is particularly useful when the two-vector approach is combined with the splice donor and acceptor approach, as the use of exon sequences will result in part of the protein being spliced out, which is generally undesirable.

The COL4A3, COL4A4, or COL4A5 transgene may encode a COL4A3, COL4A4, or COL4A5 polypeptide, or a fragment or derivative thereof.

The COL4A3, COL4A4, or COL4A5 polypeptide, or fragment or derivative thereof, may be capable of forming a collagen IV α 345 network. Suitably, about 200-300 amino acids of the fragment are removed.

In some embodiments, the COL4A3, COL4A4, or COL4A5 polypeptide is a full-length polypeptide.

Preferably, the COL4A3, COL4A4 or COL4A5 polypeptide is human. Exemplary human COL4A3 is COL4A3 with UniProtKB accession number Q01955. Example human COL4A4 is COL4A3 with UniProtKB accession number P53420. Example human COL4A5 is COL4A5 with UniProtKB accession number P29400.

Suitably, the COL4A3 peptide may comprise an amino acid sequence as set forth in SEQ ID NO:1 or a polypeptide sequence which is similar to the polypeptide sequence shown in SEQ ID NO:1 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:1 is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Suitably, the COL4A4 peptide may comprise an amino acid sequence as set forth in SEQ ID NO:2 or a polypeptide sequence substantially identical to SEQ ID NO:2 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:2 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Suitably, the COL4A5 peptide may comprise an amino acid sequence as set forth in SEQ ID NO:3 or a polypeptide sequence corresponding to seq id NO:3 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:3 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary COL4A3 amino acid sequence (SEQ ID NO: 1)

Exemplary COL4A4 amino acid sequence (SEQ ID NO: 2)

Exemplary COL4A5 amino acid sequence (SEQ ID NO: 3)

An exemplary nucleotide sequence encoding COL4A3 is NM — 000091.5. An exemplary nucleotide sequence encoding COL4A4 is NM — 000092.5. An exemplary nucleotide sequence encoding COL4A5 is NM — 000495.5.

Suitably, the COL4A3 transgene may comprise the sequence as set forth in SEQ ID NO:4 or a polynucleotide sequence substantially identical to SEQ ID NO:4 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:4 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Suitably, the COL4A4 transgene may comprise the sequence as set forth in SEQ ID NO:5 or a polynucleotide sequence substantially identical to SEQ ID NO:5 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:5 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Suitably, the COL4A5 transgene may comprise the sequence as set forth in SEQ ID NO:6 or a polynucleotide sequence substantially identical to SEQ ID NO:6 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:6 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary COL4A3 transgene sequence (SEQ ID NO: 4)

Exemplary COL4A4 transgene sequence (SEQ ID NO: 5)

Exemplary COL4A5 transgene sequence (SEQ ID NO: 6)

The COL4A3, COL4A4 or COL4A5 transgene may be codon optimized. The choice of a particular codon varies from cell to cell. This codon bias corresponds to the bias in the relative abundance of a particular tRNA in a cell type. By changing the codons in the sequence such that they are tailored to match the relative abundance of the corresponding trnas, it is possible to increase expression. For the same reason, it is possible to reduce expression by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, an additional degree of translation control may be obtained. Codon usage tables for mammalian cells (e.g., humans) as well as for a variety of other organisms are known in the art.

Regulatory sequences

Promoters

The viral vector of the present invention may comprise a promoter that promotes expression of COL4A3, COL4A4, or COL4A5 polypeptide. Suitably, the promoter may be operably linked to a COL4A3, COL4A4 or COL4A5 transgene.

Preferably, the promoter is operable in podocytes. Preferably, the promoter is capable of driving transgene expression in podocytes. Preferably, the viral vector of the invention comprises a podocyte-specific promoter. Suitably, the COL4A3, COL4A4 or COL4A5 transgene is operably linked to a podocyte-specific promoter.

As described above, the inventors have developed a minimal nephrin promoter that is shorter than known minimal nephrin promoters and surprisingly capable of driving transgene expression in podocytes. The promoter also surprisingly retains podocyte specificity. Such a minimal nephrin promoter can be used to minimize cargo size and aid in packaging of full-length COL4A3, COL4A4, or COL4A5.

The use of podocyte-specific promoters, such as the minimal nephrin promoter, allows for the specific targeting of viral vectors to podocytes (Moeller et al, 2002 picconi et al, 2014). Suitable minimal nephrin promoters include NPHS1 and podocin promoter NPHS2. This enables transgene expression to specifically target podocytes in the kidney glomerular basement membrane and minimize off-target expression. Since podocytes are terminally differentiated, non-dividing cells, they can be targeted for stable expression of the transgene and reduce or avoid the risk of any vector dilution effects. In a preferred embodiment of the invention, the promoter is NPHS1. An example of a suitable DNA sequence for the NPHS1 promoter is shown in figure 1. As with the transgene, the species of the promoter is preferably matched to the patient species. For example, human NHPS1 or human NPHS2 is commonly used in the treatment of human patients.

As used herein, a "podocyte-specific promoter" can be a promoter that preferentially promotes expression of a transgene in a podocyte. Suitably, the podocyte-specific promoter may promote higher expression of the transgene in the podocyte as compared to other cell types. For example, a podocyte-specific promoter can be a promoter that promotes a transgene expression level at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in a podocyte as compared to the expression level in other cell types.

Transgene expression can be measured by any suitable method known in the art. For example, by measuring the expression of a reporter transgene, such as GFP, operably linked to a promoter, wherein expression of the reporter transgene correlates with the promoter's ability to promote gene expression. Expression of the reporter transgene, e.g., GFP, can be determined by any suitable method, e.g., FACS. For example, podocyte-specific promoters can promote higher expression of reporter transgenes in conditionally-immortalized podocytes as compared to other cell types such as glomerular endothelial cells. Suitable podocyte cell lines will be well known to those skilled in the art, such as CIHP-1. Methods for generating immortalized podocytes will be well known to those skilled in the art. Suitable methods are described in Ni, l., et al, 2012.Nephrology,17 (6), pp.525-531.

Suitably, the promoter may be a minimal podocyte-specific promoter. The promoter may have a length of about 1.2kb or less. Suitably, the promoter has a length of about 1.18kb or less, about 1.17kb or less, about 1.16kb or less, about 1.15kb or less, about 1.14kb or less, about 1.13kb or less, about 1.12kb or less, about 1.11kb or less, or about 1.10kb or less. Suitably, the promoter has a length of about 1.15kb or less. The promoter may have a length of about 1.1kb or less. In some embodiments, the promoter has a length of about 1.1kb or less, 1.0kb or less, about 0.9kb or less, about 0.8kb or less, about 0.7kb or less, about 0.6kb or less, about 0.5kb or less, about 0.4kb or less, or about 0.3kb or less.

In some embodiments, the promoter has a length of about 0.8kb or less, about 0.7kb or less, about 0.6kb or less, about 0.5kb or less, about 0.4kb or less, or about 0.3kb or less. In some embodiments, the promoter has a length of 818bp or less. In some embodiments, the promoter has a length of 800bp or less. In some embodiments, the promoter has a length of about 0.5kb or less, about 0.4kb or less, or about 0.3kb or less. In some embodiments, the promoter has a length of about 0.3kb or less.

The promoter may have a length of about 250bp or more. In some embodiments, the promoter has a length of about 250-1100bp, 250-1000bp, 250-900bp, 250-800bp, 250-700bp, 250-600bp, 250-500bp, 250-400bp, 250-300 bp. The promoter may have a length of about 265bp or greater. In some embodiments, the promoter has a length of about 265-1100bp, 265-1000bp, 265-900bp, 265-800bp, 265-700bp, 265-600bp, 265-500bp, 265-400bp, 265-300 bp. In one embodiment, the promoter has a length of 250-300bp, 250-280bp, 255-275bp, 260-270bp, or about 265 bp. In one embodiment, the promoter has a length of 800-850bp, 800-840bp, 810-830bp, 815-825bp, or about 819 bp.

Minimal nephrin promoter

The viral vector of the invention may comprise a minimal nephrin promoter. Suitably, the minimal nephrin promoter may be operably linked to a COL4A3, COL4A4 or COL4A5 transgene.

The minimum nephrin promoter may be the minimum NPHS1 promoter. For example, the NPHS1 promoter may have a length of 1.2kb or less. The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes.

The minimal human NPHS1 promoter has been described in Moeller et al 2002J Am Soc Nephrol,13 (6): 1561-7 and Wong MA et al.2000Am J Physiol Renal Physiol,279 (6): f1027-32. This minimal NPHS1 is a 1.2kb fragment and appears to be podocyte-specific. The 1.2kb promoter region lacks the TATA box, but has recognition motifs for other transcription factors, such as PAX-2 binding elements, E-box, and GATA consensus sequences.

Suitably, the minimal nephrin promoter may comprise the sequence as set forth in SEQ ID NO:7 or a nucleotide sequence corresponding to SEQ ID NO:7 (also shown in figure 1) or consists of a variant which is at least 70% identical.

Exemplary minimal NPHS1 promoter (SEQ ID NO: 7):

suitably, the variant may be identical to SEQ ID NO:7 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical.

Suitably, the minimal nephrin promoter may comprise a sequence as set forth in SEQ ID NO:8 or a nucleotide sequence substantially identical to SEQ ID NO:8 or consists of a variant which is at least 70% identical.

Exemplary minimal NPHS1 promoter (SEQ ID NO: 8):

suitably, the variant may be identical to SEQ ID NO:8 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical.

In some embodiments, the minimal nephrin promoter comprises the amino acid sequence as set forth in SEQ ID NO:9 or a nucleotide sequence corresponding to SEQ ID NO:9 or consists of a variant which is at least 70% identical.

Exemplary minimal nephrin promoter-819 bp (SEQ ID NO: 9)

Suitably, the variant may be identical to SEQ ID NO:9 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical.

In a preferred embodiment, the minimal nephrin promoter comprises the amino acid sequence as set forth in SEQ ID NO:10 or a nucleotide sequence substantially identical to SEQ ID NO:10 or a variant consisting of at least 70% identical.

Exemplary minimal nephrin promoter-265 bp (SEQ ID NO: 10)

Suitably, the variant may be identical to SEQ ID NO:10 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical.

Suitably, the minimal nephrin promoter is derived from SEQ ID NO:8 or a variant of SEQ ID NO:8 variants having at least 70% identity. Suitably, the minimal nephrin promoter is operably linked to SEQ ID NO:8 or a variant of SEQ ID NO:8 have one or more deletions compared to a variant having at least 70% identity. Suitably, the variant may be identical to SEQ ID NO:8 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. Exemplary variants are SEQ ID NOs: 7.

suitably, the minimal nephrin promoter comprises a sequence according to SEQ ID NO:8 and has one or more deletions, e.g., one or two deletions, or a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, or consists thereof. Suitably, the minimal nephrin promoter comprises a sequence according to SEQ ID NO:8 or a nucleotide sequence having two or more deletions or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. The deletions may be of any size. Suitably, the size of the deletion is each at least 50bp, at least 100bp, at least 150bp, at least 200bp, at least 250bp, at least 300bp, at least 350bp or at least 400bp. Suitably, the size of the deletions is each 50 to 500bp, 100 to 500bp, 150 to 500bp, 200 to 500bp, 250 to 500bp, 300 to 500bp, 350 to 500bp or 400 to 500bp.

In some embodiments, the minimal nephrin promoter comprises or consists of a sequence according to SEQ ID NO:8, but wherein:

(i) Deletion of SEQ ID NO:8 from position 1 to position n1, wherein n1 is an integer from 1 to 430; and/or

(ii) Deletion of SEQ ID NO:8 from position n2 to position n3, wherein n3 ≧ n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061;

or a nucleotide sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

For example, the minimal nephrin promoter may comprise or consist of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a nucleotide sequence according to SEQ ID NO:8, but wherein:

(ii) Deletion of SEQ ID NO:8 from position n2 to position n3, wherein n3 ≧ n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061.

Suitably, n1 is an integer from 50 to 430, 100 to 430, 150 to 430, 200 to 430, 250 to 430, 300 to 430, 350 to 430 or 400 to 430. In some embodiments, n1 is an integer from 100 to 430. In some embodiments, n1=430, i.e., deleting the amino acid sequence of SEQ ID NO: position 1 to position 430 of 8.

The difference between n3 and n2 specifies the size of the deletion. Suitably, n 3. Gtoreq.n 2+49, n 3. Gtoreq.n 2+99, n 3. Gtoreq.n 2+149, n 3. Gtoreq.n 2+199, n 3. Gtoreq.n 2+249, n 3. Gtoreq.n 2+299, n 3. Gtoreq.n 2+349, n 3. Gtoreq.n 2+399, n 3. Gtoreq.n 2+449, n 3. Gtoreq.n 2+499 or n 3. Gtoreq.n 2+549. In some embodiments, n3 ≧ n2+49.

The values adopted by n2 and n3 determine the location of the deletion. Suitably, n2 and n3 are each integers of 550 to 1050, n2 and n3 are each integers of 600 to 1000, n2 and n3 are each integers of 650 to 950, n2 and n3 are each integers of 700 to 900, and n2 and n3 are integers of 750 to 850. In some embodiments, n2=508 and n3=1061, i.e., deleting the amino acid sequence of SEQ ID NO: positions 508 to 1061 of 8.

Minimum nephrin promoter region

The inventors have identified the region of the nephrin promoter driving transgene expression.

Promoters generally comprise a "core" and a "proximal" region. The "core promoter region" may comprise a transcription initiation site, an RNA polymerase binding site, and a general transcription factor binding site. The "proximal promoter region" may comprise, for example, the primary regulatory elements and specific transcription factor binding sites required to promote efficient and controllable transcription. The size and composition of the core and proximal promoter regions typically vary in a gene-specific manner. The promoter may also comprise a5 'untranslated region (5' utr) (also known as leader sequence) downstream of the core promoter region and upstream of the start codon.

The minimal nephrin promoter may be a hybrid promoter. As used herein, "hybrid promoter" includes a combination of elements derived from different promoters. For example, a hybrid promoter may comprise a proximal promoter region derived from one pre-existing promoter and a core promoter from another pre-existing promoter to achieve desired transgene expression. Muscle hybrid promoters are described in Piekarowicz, K., et al (2019), methods & clinical definition, 15, 157-169.

In some embodiments, the minimal nephrin promoter comprises (i) the amino acid sequence as set forth in SEQ ID NO:12, or a nucleotide sequence corresponding to SEQ ID NO:12 variants that are at least 70% identical. Without wishing to be bound by theory, it is believed that the sequence of SEQ ID NO:12 can provide a proximal promoter region.

Exemplary proximal promoter region (SEQ ID NO: 12)

Suitably, the variant may be identical to SEQ ID NO:12 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise SEQ ID NO:12, as set forth in SEQ ID NO: shown at 13.

Exemplary variant proximal promoter region (SEQ ID NO: 13)

In some embodiments, the minimal nephrin promoter comprises (ii) a sequence as set forth in SEQ ID NO:14, or a nucleotide sequence corresponding to SEQ ID NO:14 variants that are at least 70% identical; as shown in SEQ ID NO:15, or a nucleotide sequence corresponding to SEQ ID NO: a variant that is 15 at least 70% identical; and/or as shown in seq id NO:16, or a nucleotide sequence corresponding to SEQ ID NO:16 variants that are at least 70% identical.

In some embodiments, the minimal nephrin promoter comprises (ii) a sequence as set forth in SEQ ID NO:14, or a nucleotide sequence corresponding to SEQ ID NO:14 variants that are at least 70% identical. Without wishing to be bound by theory, it is believed that the sequence of SEQ ID NO:14 can provide a core promoter region, having at least about 70% identity thereto.

Exemplary core promoter region (SEQ ID NO: 14)

Suitably, the variant may be identical to SEQ ID NO:14 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise SEQ ID NO:14, as set forth in SEQ ID NO: shown at 17.

Exemplary variant core promoter region (SEQ ID NO: 17)

In some embodiments, the minimal nephrin promoter comprises (ii) a sequence as set forth in SEQ ID NO:15, or a nucleotide sequence corresponding to SEQ ID NO:15 variants that are at least 70% identical. Without wishing to be bound by theory, it is believed that the sequence of SEQ ID NO:15 nucleotide sequences having at least about 70% identity can provide a 5' utr.

Exemplary 5' UTR (SEQ ID NO: 15)

Suitably, the variant may be identical to SEQ ID NO:15 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise SEQ ID NO:15, as set forth in SEQ ID NO:18, respectively.

Exemplary variant 5' UTR (SEQ ID NO: 18)

In some embodiments, the minimal nephrin promoter comprises (ii) a sequence as set forth in SEQ ID NO:16, or a nucleotide sequence corresponding to SEQ ID NO:16 variants that are at least 70% identical. Without wishing to be bound by theory, it is believed that the sequence of SEQ ID NO:16 can provide a core promoter region and 5' utr.

Exemplary core promoter region and 5' UTR (SEQ ID NO: 16)

Suitably, the variant may be identical to SEQ ID NO:16 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise SEQ ID NO:16, as set forth in SEQ ID NO:19, respectively.

Exemplary variant core promoter region and 5' UTR (SEQ ID NO: 19)

In some embodiments, the minimal nephrin promoter comprises (iii) a sequence that is identical to SEQ ID NO:20 or one or more fragments thereof having at least 70% identity. Suitably, the minimal nephrin promoter comprises a sequence identical to SEQ ID NO:20 or one or more fragments thereof having at least about 70% identity immediately downstream of the proximal promoter region and/or immediately upstream of the core promoter region.

The minimal nephrin promoter may comprise a sequence identical to SEQ ID NO:20 or one or more fragments thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. The minimal nephrin promoter may comprise SEQ ID NO:20, or one or more fragments thereof.

Exemplary promoters regions of anyhow (SEQ ID NO: 20)

Suitably, the one or more fragments are (a) a 5' terminal fragment; and/or (b) a 3' terminal fragment. Suitably, the 5' end fragment may be immediately downstream of the proximal promoter region. Suitably, the 3' terminal fragment may be immediately upstream of the core promoter region. For example, the minimal nephrin promoter may include:

(a) And SEQ ID NO:20 has at least 70% of the nucleotide sequence from position 1 to x; and/or

(b) And SEQ ID NO:20 from positions y to 554 of at least 70% identity;

wherein x and y are integers, and y > x.

The amino acid sequence of SEQ ID NO: the segments of 20 may be of any length. Suitably, the fragments may have a length of about 500bp or less, 450bp or less, 400bp or less, 350bp or less, 300bp or less, 250bp or less, 200bp or less, 150bp or less, 100bp or less, 50bp or less, 40bp or less, 30bp or less, 20bp or less, or 10bp or less.

In some embodiments, the minimal nephrin promoter does not comprise SEQ ID NO:20.

in some embodiments, the minimal nephrin promoter comprises (iv) a sequence that is identical to SEQ ID NO:21 or a fragment thereof having a nucleotide sequence of at least 70% identity. Suitably, the minimal nephrin promoter comprises a sequence identical to SEQ ID NO:21 or a fragment thereof having at least about 70% identity to the nucleotide sequence immediately upstream of the promoter region.

The minimal nephrin promoter may comprise a sequence identical to SEQ ID NO:21 or a fragment thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. The minimal nephrin promoter may comprise SEQ ID NO:21, or one or more fragments thereof.

Exemplary optional upstream promoter region (SEQ ID NO: 21)

Suitably, the fragment is a 3' terminal fragment. For example, the minimal nephrin promoter may comprise a sequence identical to SEQ ID NO:21, wherein z is an integer, to 430, a nucleotide sequence having at least 70% identity.

SEQ ID NO: the segments of 21 may be of any length. Suitably, the fragment may have a length of about 400bp or less, 350bp or less, 300bp or less, 250bp or less, 200bp or less, 150bp or less, 100bp or less, 50bp or less, 40bp or less, 30bp or less, 20bp or less, or 10bp or less.

In some embodiments, the minimal nephrin promoter does not comprise SEQ ID NO:21.

in some embodiments, the minimal nephrin promoter comprises or consists of, from 5 'to 3':

(i) And SEQ ID NO:12 a nucleotide sequence having at least 70% identity;

(iii) Optionally with SEQ ID NO:20 or one or more fragments thereof having at least 70% identity; and

(ii) And SEQ ID NO:14, a nucleotide sequence having at least 70% identity to SEQ ID NO:15, and/or a nucleotide sequence having at least 70% identity to SEQ ID NO:16, or a nucleotide sequence having at least 70% identity thereto.

(i) And SEQ ID NO:12 a nucleotide sequence having at least 70% identity;

(iii) Optionally (a) a sequence identical to SEQ ID NO:20 has a nucleotide sequence of at least 70% identity to the 5' fragment; and/or (b) a variant of SEQ ID NO:20 has a nucleotide sequence of at least 70% identity to the 3' fragment; and

(ii) And SEQ ID NO:14, a nucleotide sequence having at least 70% identity to SEQ ID NO:15, and/or a nucleotide sequence having at least 70% identity to SEQ ID NO:16 with at least 70% identity.

Minimal nephrin promoter element

The present inventors have identified functional elements of the nephrin promoter that drive transgene expression.

The minimum nephrin promoter may comprise one or more of the following elements: (a) a retinoic acid receptor binding site; (b) a WT1 binding site; (c) an enhancer cassette; (d) a transcription factor binding region; and (e) a transcription start site.

Suitably, the minimal nephrin promoter comprises all of the following elements: (ii) (a) a retinoic acid receptor binding site; (b) a WT1 binding site; (c) an enhancer cassette; (d) a transcription factor binding region; and (e) a transcription start site.

Retinoic Acid Receptor (RAR) binding site refers to a polynucleotide sequence capable of binding RAR α, RAR β, and/or RAR γ. The RAR binding site may comprise an amino acid sequence as set forth in SEQ ID NO:22 or a nucleotide sequence substantially identical to SEQ ID NO:22 by one or two substitutions, deletions or insertions. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the RAR binding site retains at least one of its endogenous functions.

Exemplary RAR binding site (SEQ ID NO: 22)

The WT1 binding site refers to a polynucleotide sequence capable of binding a zinc finger polypeptide encoded by Wilms tumor suppressor gene WT 1. The WT1 binding site may comprise the sequence set forth as SEQ ID NO:23 or a nucleotide sequence substantially identical to SEQ ID NO:23 to a nucleotide sequence having or consisting of one, two or three substitutions, deletions or insertions. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the WT 1-binding region retains at least one of its endogenous functions.

Exemplary WT1 binding site (SEQ ID NO: 23)

Enhancer cassettes refer to DNA response elements found in some eukaryotes that serve as protein binding sites. The enhancer cassette may comprise a sequence as set forth in SEQ ID NO:24 or a nucleotide sequence substantially identical to SEQ ID NO:24 to a nucleotide sequence having or consisting of one or two substitutions, deletions or insertions. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the enhancer cassette retains at least one of its endogenous functions.

Exemplary enhancer cassette (SEQ ID NO: 24)

(a) A retinoic acid receptor binding site; (b) a WT1 binding site; (c) One or more of the enhancer cassettes may be present in the proximal promoter region. Suitably, (a) a retinoic acid receptor binding site; (b) a WT1 binding site; (c) Each of the enhancer cassettes is present in the proximal promoter region.

In some embodiments, one or more of the following elements are present in (i) a sequence that is identical to SEQ ID NO:12 in a nucleotide sequence having at least 70% identity: (a) generating a signal at a position corresponding approximately to SEQ ID NO: a RAR binding site at position 7 to position 13 of position 12; (b) generating a nucleotide sequence at a position corresponding approximately to SEQ ID NO:12 at position 14 to position 30; (c) converting the amino acid sequence at a position approximately corresponding to SEQ ID NO:12 at position 49 to 53. In some embodiments, each of the elements is present in (i) a nucleotide sequence that is identical to SEQ ID NO:12 in a nucleotide sequence having at least 70% identity.

In some embodiments, one or more of the following nucleotide sequences are present in (i) a nucleotide sequence that is identical to SEQ ID NO:12 in a nucleotide sequence having at least 70% identity: (a) a sequence corresponding to SEQ ID NO: GGGGTCA at position 7 to position 13 of 12; (b) a sequence corresponding to SEQ ID NO:12 CGGAGGCTGGGGAGGCA at position 14 to position 30; (c) a sequence corresponding to SEQ ID NO:12 at position 49 to position 53. In some embodiments, each of the nucleotide sequences is present in (i) a nucleotide sequence that is identical to SEQ ID NO:12 in a nucleotide sequence having at least 70% identity.

Suitably, the minimal nephrin promoter may comprise a transcription factor binding region comprising a sequence as set forth in SEQ ID NO:25 or a nucleotide sequence substantially identical to SEQ ID NO:25 to a nucleotide sequence having or consisting of 1, 2, 3, 4 or 5 substitutions, deletions or insertions. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the transcription factor binding region retains at least one of its endogenous functions.

Exemplary transcription factor binding region (SEQ ID NO: 25)

Other suitable transcription factor binding regions will be well known to those skilled in the art. For example, other suitable transcription factor binding regions include TACGAT (SEQ ID NO: 36), TATAAT (SEQ ID NO: 37), GATACT (SEQ ID NO: 38), TATGAT (SEQ ID NO: 39), and TATGTT (SEQ ID NO: 40).

Suitably, the minimal nephrin promoter may comprise a transcription start site comprising or consisting of an "AG" dinucleotide.

Suitably, the transcription factor binding site is operably linked to a transcription initiation site. Suitably, the transcription factor binding site may be located directly upstream of the transcription initiation site. Without wishing to be bound by theory, it is believed that the transcription factor binding site and the transcription start site may provide a core promoter region.

Suitably, the minimal nephrin promoter may comprise a 5' untranslated region. The 5' untranslated region may comprise a nucleotide sequence identical to SEQ ID NO:15, or consists of a nucleotide sequence having at least about 70%, 80%, 90%, 95%, or 99% sequence identity. The 5' untranslated region may comprise SEQ ID NO:15 or a polypeptide consisting of SEQ ID NO:15, the components are mixed.

Suitably, the 5' untranslated region is operably linked to the transcription start site. Suitably, the 5' untranslated region may be located directly downstream of the transcription start site.

(i) And SEQ ID NO:12, wherein each of the following elements is present: (a) generating a signal at a position corresponding approximately to SEQ ID NO: a RAR binding site at position 7 to position 13 of position 12; (b) a nucleic acid sequence at a position corresponding approximately to SEQ ID NO:12 at position 14 to position 30; (c) converting the amino acid sequence at a position corresponding to about SEQ ID NO:12, an enhancer sub-box at the position 49 to position 53;

(ii) And SEQ ID NO:14, a nucleotide sequence having at least 70% identity to SEQ ID NO:15, or a nucleotide sequence having at least 70% identity to SEQ ID NO:16 with at least 70% identity.

Exemplary minimal nephrin promoters

In some embodiments, the minimal nephrin promoter comprises the amino acid sequence as set forth in SEQ ID NO:9, or a nucleotide sequence corresponding to SEQ ID NO:9 at least 70% identical and wherein the promoter has a length of about 1.1kb or less.

In some embodiments, the minimal nephrin promoter consists of the amino acid sequence as set forth in SEQ ID NO:9 or a nucleotide sequence substantially identical to SEQ ID NO:9 variant compositions that are at least 70% identical.

Suitably, the variant may be identical to SEQ ID NO:9 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise a sequence as set forth in SEQ ID NO:34, SEQ ID NO:9 or consists thereof.

Exemplary minimal nephrin promoter variant-819 bp (SEQ ID NO: 34)

In some embodiments, the minimal nephrin promoter comprises the amino acid sequence as set forth in SEQ ID NO:10 or a nucleotide sequence substantially identical to SEQ ID NO:10 or consists of a variant which is at least 70% identical.

In some embodiments, the minimal nephrin promoter comprises the amino acid sequence as set forth in SEQ ID NO:10, or a nucleotide sequence corresponding to SEQ ID NO:10 variants that are at least 70% identical and wherein the promoter has a length of about 1.1kb or less.

In some embodiments, the minimal nephrin promoter consists of the amino acid sequence as set forth in SEQ ID NO:10 or a nucleotide sequence corresponding to SEQ ID NO:10 variant compositions that are at least 70% identical.

Suitably, the variant may be identical to SEQ ID NO:10 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. The minimal nephrin promoter may comprise the sequence as set forth in SEQ ID NO:35, SEQ ID NO:10 or consists thereof.

Exemplary minimal nephrin promoter variant-265 bp (SEQ ID NO: 35)

Minimal podocin promoter

The viral vectors of the invention may comprise a minimal podocin promoter. Suitably, the minimal podocin promoter may be operably linked to a COL4A3, COL4A4 or COL4A5 transgene.

The minimal podocin promoter may be a minimal NPHS2 promoter. For example, the NPHS2 promoter may have a length of 0.6kb or less. The NPHS2 gene encodes podocin that is selectively expressed in podocytes.

The minimal human NPHS2 promoter is described in Oleggini R, et al, 2006.Gene expr.13 (1): 59-66. This minimal NPHS2 is a 630bp fragment, which has been shown to be expressed in podocytes in vitro.

Suitably, the minimal podocin promoter may comprise a nucleotide sequence as set forth in SEQ ID NO:11 or a nucleotide sequence corresponding to SEQ ID NO:11 or consists of a variant which is at least 70% identical.

Exemplary minimal NPHS2 promoter (SEQ ID NO: 11):

suitably, the variant may be identical to SEQ ID NO:11 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical.

Other promoters

Other non-podocyte-specific promoters for use in the present invention will be well known to those skilled in the art. In some embodiments, the promoter may have a length of about 300bp or less. In some embodiments, the promoter has a length of about 290bp or less, 280bp or less, 270bp or less, 260bp or less, 250bp or less, 240bp or less, 230bp or less, 220bp or less, 210bp or less, or 200bp or less. The use of promoters of about 300bp or less in length may help package COL4A3, COL4A4, and COL4A5 transgenes into AAV vectors in their full-length form.

Exemplary promoters having a length of about 300bp or less are described in Wang, D., et al, 1999.Gene therapy,6 (4), PP.667-675. Wang et al describe four short promoters with significantly higher activity than AAV ITRs alone and sizes from 102bp to 200bp. These promoters are AAV-P5 (150 bp), SV40e (200 bp), TK1 (110 bp), and a second TK promoter (TK 2) (102 bp) with an additional 10bp deletion between the distal and proximal elements.

Woodchuck hepatitis posttranscriptional regulatory element

The viral vector may additionally comprise woodchuck hepatitis post-transcriptional regulatory element (WPRE). Suitably, the WPRE may be operably linked to a COL4A3, COL4A4 or COL4A5 transgene. WPRE is a DNA sequence that creates a tertiary structure that enhances expression when transcribed. Inclusion of the WPRE can increase expression of the transgene delivered by the vector. The WPRE sequence may be mutated to reduce carcinogenicity without significant loss of RNA enhancing activity (Schambach et al, 2005, incorporated herein by reference). One example of a suitable WPRE sequence is shown in figure 2.

Suitably, the WPRE may comprise the amino acid sequence as set out in SEQ ID NO:26, or a nucleotide sequence corresponding to SEQ ID NO:26 (also shown in figure 2) or consists of a variant which is at least 70% identical.

Exemplary WPRE (SEQ ID NO: 26)

Suitably, the variant may be identical to SEQ ID NO:26 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

In some embodiments, the viral vectors of the invention do not comprise a WPRE sequence.

Protein tag

The COL4A3, COL4A4 or COL4A5 transgene may comprise a protein tag, such as a Hemagglutinin (HA) tag. HA can be used as an epitope tag and HAs been shown not to interfere with the biological activity or biodistribution of the protein to which it HAs been added. Protein tags can facilitate detection, isolation and purification of transgenes. Other suitable protein tags may include the Myc tag, the polyhistidine tag, and the flag tag.

In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene comprises one or more flag tags. In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene comprises three flag tags.

Kozak sequence

The viral vector may additionally comprise a Kozak sequence between the promoter and the COL4A3, COL4A4 or COL4A5 transgene. The Kozak sequence is known to play a major role in the initiation of the translation process and may therefore enhance expression of the COL4A3, COL4A4 or COL4A5 transgene. Suitable Kozak sequences will be well known to those skilled in the art.

Suitably, the Kozak sequence may comprise a sequence as set forth in SEQ ID NO:27 or a nucleotide sequence corresponding to SEQ ID NO:27 or consists of a variant which is at least 65% identical.

Exemplary Kozak sequence (SEQ ID NO: 27)

Suitably, the variant may be identical to SEQ ID NO:27 are at least 75%, at least 85%, or at least 90% identical.

In some embodiments, the viral vectors of the invention do not comprise a Kozak sequence.

Polyadenylation signal

The viral vector may additionally comprise a polyadenylation signal, such as the bovine growth hormone (bGH) polyadenylation signal, for example, as shown in fig. 3. Suitably, the polyadenylation signal may be operably linked to the COL4A3, COL4A4 or COL4A5 transgene. Polyadenylation is the addition of a poly (A) tail to messenger RNA. The poly (A) tail consists of multiple adenosines monophosphate; in other words, it is a stretch of RNA with only adenine bases. The poly (A) tail is important for nuclear export, translation and stability of mRNA. Thus, inclusion of a polyadenylation signal may enhance expression of a COL4A3, COL4A4, or COL4A5 transgene.

Suitable polyadenylation signals include the early SV40 polyadenylation signal (SV 40 pA), the chicken beta-globin polyadenylation signal, the bovine growth hormone polyadenylation signal (bGH), or the soluble neuropilin-1 polyadenylation signal. In some embodiments, the polyadenylation signal is the early SV40 polyadenylation signal (SV 40 pA) or the chicken β -globin polyadenylation signal. Preferably, the polyadenylation signal is the early SV40 polyadenylation signal (SV 40 pA).

Suitably, the polyadenylation signal may comprise a sequence as set forth in SEQ ID NO:28 or a nucleotide sequence corresponding to SEQ ID NO:28 (also shown in figure 3) or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:28 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary bGH poly (A) signal sequence (SEQ ID NO: 28):

suitably, the polyadenylation signal may comprise a sequence as set forth in SEQ ID NO:29 or a nucleotide sequence corresponding to SEQ ID NO:29 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:29 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary of the soluble neuropilin-1 polyadenylation signal (SEQ ID NO:29 XX):

suitably, the polyadenylation signal may comprise a sequence as set forth in SEQ ID NO:30 or a nucleotide sequence corresponding to SEQ ID NO: a variant which is at least 70% identical or consists thereof. Suitably, the variant may be identical to SEQ ID NO:30 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary SV40pA signal sequence (SEQ ID NO: 30):

suitably, the polyadenylation signal may comprise a sequence as set forth in SEQ ID NO:41 or a nucleotide sequence corresponding to SEQ ID NO:41 or consists of a variant which is at least 70% identical. Suitably, the variant may be identical to SEQ ID NO:41 are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical.

Exemplary Chicken beta-globin polyadenylation Signal (SEQ ID NO: 41)

Inverted terminal repeat sequence

The viral vector may additionally comprise Inverted Terminal Repeat (ITR) sequences at either end of the vector. For example, the support structure may be, in order: ITR-promoter-transgene (with optional protein tag) -optional WRPE-polyadenylation signal-ITR.

The ITR can act as a promoter (Flotte, T.R., et al.1993.Journal if Biological Chemistry,268 (5), pp.3781-3790).

Typically, the AAV genome will comprise at least one Inverted Terminal Repeat (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes of different serotypes, or may be chimeric or mutated ITRs. Preferred mutant ITRs are those with a trs (terminal resolution site) deletion. This deletion allows the genome to continue to replicate to generate a single-stranded genome containing the coding and complementary sequences, i.e., a self-complementary AAV genome. This allows bypassing DNA replication in the target cell, thus enabling accelerated transgene expression. However, the maximum packaging capacity of scAAV decreased. Suitably, the AAV genome is not a scAAV genome.

The AAV genome may comprise one or more ITR sequences from any naturally derived AAV serotype, isolate or clade or variant thereof. The AAV genome may comprise at least one, such as two AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. Suitably, the AAV genome may comprise at least one, such as two AAV2 ITRs.

One or more ITRs are preferably included to help the AAV vector form concatemers in the host cell nucleus, for example after conversion of single stranded vector DNA to double stranded DNA by the action of a host cell DNA polymerase. The formation of this episomal concatemer protects the AAV vector during the life cycle of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

Suitably, the ITR element will be the only sequence in the derivative that is retained from the native AAV genome. Derivatives preferably do not include rep and/or cap genes of the natural genome as well as any other sequences of the natural genome. This is preferred for the reasons mentioned above and is also preferred for reducing the likelihood of integration of the vector into the host cell genome. Furthermore, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) in addition to the transgene within the vector.

Variants, derivatives, analogs, homologs and fragments

In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses variants, derivatives, homologues and fragments thereof.

In the context of the present invention, a "variant" of any given sequence is a sequence in which the particular sequence of residues (whether amino acid residues or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences may be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one residue present in the naturally-occurring polypeptide or polynucleotide. For example, a variant promoter sequence retains at least some level of activity and specificity of the promoter sequence from which it is obtained.

The term "derivative" as used herein in relation to a protein or polypeptide of the invention includes any substitution, variation, modification, substitution, deletion and/or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

In general, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs.

The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues which result in silent changes and produce functionally equivalent proteins. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine, and tyrosine.

Conservative substitutions may be made, for example, according to the following table. The amino acids in the same block in the second column, preferably in the same row in the third column, may be substituted for each other:

as used herein, the term "homologue" refers to a variant having a certain homology to a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" may be equated with "identity".

In this context, homologous sequences are understood to include amino acid sequences which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the target sequence. Typically, the homologue will comprise the same active site as the amino acid sequence of interest, etc. In the context of the present invention, homology is preferably expressed in terms of sequence identity, although homology may also be considered in terms of similarity (i.e. amino acid residues with similar chemical properties/functions).

In this context, homologous sequences are understood to include nucleotide sequences which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the target sequence. In the context of the present invention, homology is preferably expressed in terms of sequence identity, although homology may also be considered in terms of similarity.

Preferably, reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having said percent identity over the entire length of the referenced SEQ ID NO.

Homology comparisons can be performed by eye, or more commonly, with the aid of ready-made sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

Percent homology within a contiguous sequence can be calculated, i.e., one sequence is aligned with another sequence and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is referred to as a "gapless" alignment. Typically, such gap-free alignments are performed only over a relatively small number of residues.

Although this is a very simple and consistent approach, it does not take into account, for example, that in other identical sequence pairs, an insertion or deletion of one of the amino acid or nucleotide sequences may result in subsequent residues or codons not being aligned, and thus may result in a substantial reduction in percent homology when performing global alignment. Thus, most sequence comparison methods aim to produce optimal alignments, taking into account possible insertions and deletions, without unduly penalising the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximise local homology.

However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, and thus, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting a higher correlation sequence between the two being compared, will achieve a higher score than a sequence with many gaps. The "affine gap cost" is generally used to charge a relatively high cost for the presence of a gap, while charging a small penalty for each subsequent residue in the gap. This is the most commonly used vacancy scoring system. High gap penalties will of course result in an optimized alignment with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is preferred to use default values. For example, when using the GCG Wisconsin Bestfit software package, the default gap penalty for amino acid sequences is gap-12 and each extension-4.

Therefore, calculating the maximum percent homology first requires producing an optimal alignment while considering gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; devereux et al (1984) Nucleic Acids Research 12. Examples of other software that can be used for sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al (1999) supra-Ch.18), FASTA (Atschul et al, (1990) J.mol.biol.403-410), EMBOSS Needle (Madeira, F., et al, 2019.Nucleic acids research,47 (W1), pp.W636-W), and GENEWORKS comparison kit. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al (1999) supra, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, can also be used to compare protein and nucleotide Sequences (FEMS Microbiol. Lett. (1999) 174 (2): 247-50.

Although the final percent homology can be measured in terms of identity, the alignment process itself is generally not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A common example of such a matrix is the BLOSUM62 matrix (the default matrix of the BLAST suite of programs). GCG Wisconsin programs typically use public default values or custom symbol comparison tables (if provided) (for more detail, see user manual). For some applications, it is preferable to use a common default value for the GCG package, or in the case of other software, a default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible to calculate the percent homology, preferably the percent sequence identity. The software typically takes this as part of the sequence comparison and generates a numerical result. Percent sequence identity can be calculated as the number of identical residues as a percentage of the total residues in the referenced SEQ ID NO.

A "fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

Such variants, derivatives, homologues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where an insertion is to be made, 5 'and 3' flanking regions encoding the inserted synthetic DNA and the naturally occurring sequences corresponding to either side of the insertion site may be prepared. The flanking regions will contain convenient restriction sites corresponding to those in the naturally occurring sequence, so that the sequence may be cleaved with an appropriate enzyme and synthetic DNA ligated into the nick. The DNA is then expressed according to the invention to produce the encoded protein. These methods are merely illustrative of the numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

Cells

In one aspect, the invention provides a cell comprising a viral vector of the invention. The cell may be an isolated cell. The cell may be a human cell, suitably an isolated human cell.

Viral vectors can be introduced into cells using a variety of techniques known in the art, such as transfection, transduction, and transformation. Suitably, the vector of the invention is introduced into the cell by transfection or transduction.

The cells may be of any cell type known in the art.

Suitably, the cell may be a producer cell. The term "producer cell" includes a cell that produces viral particles following transient transfection, stable transfection, or vector transduction of all elements necessary for production of the viral particles, or any cell engineered to stably contain elements necessary for production of the viral particles. Suitable producer cells will be well known to those skilled in the art. Suitable producer cell lines include HEK293 (e.g. HEK 293T), heLa and a549 cell lines.

Suitably, the cell may be a packaging cell. The term "packaging cell" includes cells that contain some or all of the elements necessary for packaging of infectious recombinant virus. The packaging cell may lack the recombinant viral vector genome. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins. Cells containing only some of the elements required for the production of enveloped virus particles can be used as intermediate reagents for the generation of virus particle producing cell lines by the subsequent steps of transient transfection, transduction or stable integration of each additional required element. These intermediate reagents are encompassed by the term "packaging cells". Suitable packaging cells will be well known to those skilled in the art.

Suitably, the cell may be a kidney cell or a glomerular cell, for example a podocyte. Suitably, the cell may be an immortalised kidney cell or a glomerular cell, for example an immortalised podocyte. Suitable podocyte cell lines will be well known to those skilled in the art, such as CIHP-1. Methods for generating immortalized podocytes will be well known to those skilled in the art. Suitable methods are described in Ni, l., et al, 2012. Neuroprology, 17 (6), pp.525-531.

As noted above, although the cells are described with reference to viral vectors, it will be appreciated that viral vector gene therapy may alternatively be used.

Methods of treating or preventing Alport syndrome

In one aspect, the invention provides a viral vector, a cell or a pharmaceutical composition according to the invention for use as a medicament.

In one aspect, the invention provides the use of a viral vector, a cell or a pharmaceutical composition according to the invention for the preparation of a medicament.

In one aspect, the invention provides a method of administering a viral vector, cell or pharmaceutical composition according to the invention to a subject in need thereof.

In one aspect, the invention provides a viral vector, cell or pharmaceutical composition according to the invention for use in the prevention or treatment of Alport syndrome.

In one aspect, the invention provides the use of a viral vector, cell or pharmaceutical composition according to the invention in the manufacture of a medicament for the prevention or treatment of Alport syndrome.

In one aspect, the invention provides a method of preventing or treating Alport syndrome comprising administering a viral vector, cell or pharmaceutical composition according to the invention to a subject in need thereof.

Therapies targeting the podovirus COL4A3, COL4A4 or COL4A5 gene can alter and at least partially normalize glomerular basement membrane in AS patients. The structural effect of the constructs on the glomerular basement membrane can be tested in vitro using the human spheroid model of wild type and Alport syndrome podocytes. The spheroid model can be examined for changes in the composition of the glomerular basement membrane. Functional testing can also be performed using nephrons on a chip model that includes co-culturing glomerular endothelial cells and podocytes on one side of a channel to form a mature glomerular basement membrane that can be used to measure protein permeability through the channel. The constructs can be tested in mouse α 3 or α 5 KO mice or α 4 spontaneous mouse mutants. The construct can be administered by tail vein injection, and efficacy will be measured by proteinuria level and survival.

Viral vector gene therapy of the invention can be used to treat or prevent Alport Syndrome (AS). Patients with AS often exhibit hematuria, possibly progressing to proteinuria. Hematuria can be determined by observing the presence of red blood cells in urine under a microscope. Basal microalbuminuria levels below 30 mg/day are generally considered to be non-pathological. Levels from about 30 mg/day to about 300 mg/day are referred to as microalbuminuria, which is considered pathological. Albumin levels above 300 mg/day are termed macroalbuminuria, and proteinuria levels above 3.5 g/day are considered kidney disease-range proteinuria. Patients treated with the viral vectors of the invention may have hematuria, microalbuminuria, macroalbuminuria or nephrotic range proteinuria.

Treating a patient prior to the onset of proteinuria can slow or prevent the progression of proteinuria, thereby delaying or preventing end-stage renal failure. Patients with proteinuria in the renal range can also be treated. Protein urine levels should be gradually reduced after gene therapy treatment due to the transgenic alteration and normalization or repair of the collagen IV α 345 network of glomerular basement membrane.

The patient may additionally or alternatively detect a pathogenic variant of COL4A3, COL4A4 or COL4A5 as positive. The pathogenic variants of COL4A3 and COL4A4 may be heterozygotes (autosomal dominant) or biallels (autosomal recessive). The pathogenic variant in COL4A5 is hemizygous or heterozygous (X-linked). The patient is preferably treated with one or more viral vectors comprising a transgene corresponding to a gene of the patient having a pathogenic variant. For example, a patient with a pathogenic COL4A5 variant may be treated with a viral vector comprising a COL4A5 transgene. A patient may detect two or more pathogenic variants positive for COL4A3, COL4A4, or COL4A5. Such patients may be treated with two or more viral vectors comprising different transgenes, i.e., each viral vector comprises a transgene corresponding to a gene for which the patient has a pathogenic variant.

In particular, patients may have X-linked AS, which is often associated with pathogenic variants of COL4A5.

As used herein, the term "patient" may include any mammal, including a human. The patient may be an adult or a pediatric patient, such as a neonate or an infant. The patient may be male or female. The patient may be a male patient, particularly a juvenile male patient, suffering from X chromosome-linked AS.

The viral vectors, cells or pharmaceutical compositions according to the invention may be administered parenterally, for example intravenously or by infusion techniques. The carrier, cells or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably at a pH of 3 to 9). The pharmaceutical compositions may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

The viral vector, cell, or drug may be administered systemically, such as by intravenous injection.

The viral vector, cell or pharmaceutical composition according to the invention may be administered locally, e.g. by targeted administration to the kidney. Suitably, the viral vector, cell or pharmaceutical composition may be administered by injection into the renal artery or by ureteral or subcapsular injection. In an embodiment of the invention, the viral vector may be administered by injection into the renal artery. In an alternative embodiment of the invention, the viral vector may be administered retrogradely, e.g., via the ureter using a catheter.

The viral vector, cell or pharmaceutical composition may be administered as a single dose, in other words, subsequent doses of the vector may not be required. Where repeated doses are required, different virus serotypes may be used in the vector. For example, the vector used in the first dose may comprise AAV-LK03 or AAV-3B, while the vector used in the subsequent doses may comprise AAV 2/9.

The viral vector, cell or pharmaceutical composition may be administered in different doses (e.g., measured as vector genome (vg)/kg). In any event, the physician will determine the actual dosage which will be most suitable for any individual subject and will vary with the age, weight and response of the particular subject. In general, however, for the AAV vectors of the invention, 10 can be administered ¹⁰ To 10 ¹⁴ vg/kg or 10 ¹¹ To 10 ¹³ Dose of vg/kg.

Optionally, the viral vector, cell or pharmaceutical composition may be administered in combination with temporary immunosuppression of the patient, e.g., by administering the viral vector concurrently with or after oral steroid therapy. Immunosuppression may be required prior to and/or during gene therapy treatment to suppress the patient's immune response to the vector. However, the AAV capsid is only transiently present in the transduced cell, as it is not encoded by the vector. Thus, the capsid is gradually degraded and cleared, which means that a short-term immunomodulating regimen that blocks the immune response to the capsid until the capsid sequences are cleared from the transduced cells can allow for long-term expression of the transgene. Thus, immunosuppression may be required for a period of about six weeks after administration of gene therapy.

The viral vector, cell or pharmaceutical composition may additionally or alternatively be administered in combination with a renin-angiotensin treatment strategy, such as an Angiotensin Converting Enzyme (ACE) inhibitor, an aldosterone antagonist (e.g., spironolactone), or an Angiotensin Receptor Blocker (ARB).

Pharmaceutical composition

The viral vector may be administered in the form of a pharmaceutical composition. In other words, the viral vector may be combined with one or more pharmaceutically acceptable carriers, diluents and/or excipients. Suitable pharmaceutical compositions are preferably sterile.

Acceptable carriers, diluents and excipients for therapeutic use are well known in the pharmaceutical arts. The choice of pharmaceutically acceptable carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may contain any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent as a carrier, excipient or diluent or in addition thereto.

Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides and diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

The pharmaceutical composition may further comprise one or more other therapeutic agents.

The invention further encompasses the use of kits comprising the viral vectors, cells and/or pharmaceutical compositions of the invention. Preferably, the kit is for use in a method and is for use as described herein, for example a method of treatment as described herein. Preferably, the kit includes instructions for use of the kit components.

As noted above, while methods of treating or preventing Alport syndrome have been described by reference to viral vectors, it will be appreciated that viral vector gene therapy may alternatively be used.

Brief Description of Drawings

FIG. 1 shows an exemplary DNA sequence of the minimal human nephrin promoter (NPHS 1).

Figure 2 shows an exemplary DNA sequence of the WPRE sequence.

FIG. 3 shows an exemplary DNA sequence of the bGH poly (A) signal sequence.

Fig. 4 shows an exemplary AAV transfer plasmid comprising COL4A3, COL4A4, and COL4A5 coupled to a mini nephrin promoter.

(A)pAAV.265.

Is a schematic diagram ofAn AAV plasmid of COL4A3 coupled to a mini nephrin promoter. The SmaI sites are shown, and the following fragments are expected after restriction with SmaI: 1.6238bp, 2.2753bp, 3.56bp, 4.11bp and 5.11bp. (B) pAAV.265.

Schematic of (a), this is an AAV plasmid comprising COL4A4 coupled to a mini nephrin promoter. The SmaI site is shown, and the following fragments are expected after restriction with SmaI: 1.4052bp, 2.2753bp, 3.2224bp, 4.56bp, 5.11bp and 6.11bp. (C) pAAV.265.

Schematic representation of an AAV plasmid comprising COL4A5 coupled to the mini nephrin promoter. The SmaI site is shown, and the following fragments are expected after restriction with SmaI: 1.4272bp, 2.2753bp, 3.2032bp, 4.56bp, 5.11bp and 6.11bp. (D) restriction digestion with SmaI. MW =1Kb DNA ladder,

lanes

1, 2 and 3 correspond to digests of the plasmids shown in (a), (B) and (C), respectively. (E) shows a schematic representation of restriction digestion.

FIG. 5 shows podocytes transduced with AAV.COL4.nephrin265.Sv40 virus

(A) Immunoprecipitation experiments of FLAG-tagged full-length Col4a3 (LK 03) or Col4a5 (LK 03) were performed in human differentiated CiPodocyte (conditional immortalization) pulled with anti-FLAG antibody. The anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human FLAG IgG was used as a control. (B) Western blot of protein lysates showing the expression levels of Col4a3 (LK 03 capsid serotype), col4a5 (LK 03) and Col4a5 (2/9 capsid serotype) in human or mouse differentiated CiPodocyte. Uninfected human and mouse cipodocytes were used as controls. (C) Confocal images showing immunofluorescent staining of transduced Col4a5 and F-actin in human wild-type CiPodocyte/Col4a5 xFlag AAV CiPodocyte. Col4a5 was present at cytosolic levels in human differentiated podocytes infected with Col4a53xFlag AAV virus compared to the wild type counterpart.

FIG. 6 shows a schematic diagram of the minimal nephrin promoter

(A) The length of the full-length nephrin promoter is 1249bp (excluding the initiation codon), and is hereinafter referred to as 'FL' nephrin promoter. (B) An exemplary minimal nephrin promoter with a 5' region deleted is 819bp in length (excluding the start codon), hereinafter referred to as the "midi" nephrin promoter. (C) An exemplary minimal nephrin promoter with deletion of the 5' region and deletion of the central region is 265bp in length (excluding the start codon), hereinafter referred to as the "mini" nephrin promoter. (D) the following regions of the nephrin promoter are indicated: (ii) a Retinoic Acid Receptor (RAR) binding site, (iii) a WT1 binding site, (iv) a transcription factor binding region, and (v) a transcription initiation site.

FIG. 7 shows a schematic of a lentiviral vector comprising GFP operably coupled to a midi nephrin promoter

(A) The pACE _ hNPHS1 promoter was used as a template to introduce BamH1 and Cla1 restriction sites. (B) The vector was finally constructed, containing GFP operably coupled to the midi nephrin promoter.

FIG. 8 shows a schematic of a lentiviral vector comprising GFP operably coupled to a mini nephrin promoter

(A) The pACE _ hNPHS1 promoter was used as a template for PCR and two portions of the promoter were gel extracted. (B) The final vector was constructed containing GFP operably coupled to the mini nephrin promoter.

FIG. 9 shows expression of GFP in ciPodocyte after transduction with lentiviral vector

Human CiPodocyte stably expressing the GFP-tagged nephrin promoter was generated using a lentiviral approach. GFP expression was observed by fluorescence microscopy. (A) untransduced CiPodocyte. (B) Stably express CiPodocyte of mini nephrin promoter with GFP label. (C) Stably express CiPodocyte of FL nephrin promoter with GFP label.

FIG. 10 shows GFP expression in ciPodocyte differentiated after transduction with lentiviral vector

Lentiviral vectors comprising GFP operably linked to a nephrin promoter were transduced into differentiated conditionally-immortalized podocytes (ciPodocyte). Immunoprecipitation (IP) was used to detect GFP expression.

Figure 11 shows human glomerular cells transduced with the lentivirus-gfp. Nephrin promoter (min or 265)

FACS analysis demonstrated median GFP fluorescence (AFU) for all viable single peaks of conditionally-immortalized human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte analyzer. Untransduced cells (cell control) were compared to cells transduced with lentiviral constructs carrying a GFP expression cassette controlled by either full-length human nephrin promoter (hnpsh 1. GFP) or mini-human nephrin promoter (265. GFP). All cells were allowed to differentiate for 10 days, trypsinized (100 uL) and diluted in PBS, 2% FBS, 1: 1000 DRAQ7 (150 uL). Data and error bars represent 3 technical replicates (100 uL, > 2500 cells). + -. SEM.

Examples

Alport syndrome is a disease affecting the glomerular basement membrane collagen alpha 3 alpha 4 alpha 5 (IV) network, lacking glomerular specific therapeutic strategies. The current primary treatment is for elevated blood pressure. Elevated glomerular filtration rate and microalbuminuria are early indicators of Alport syndrome, both related to changes in the glomerular basement membrane. Collagen α 3 α 4 α 5 (IV) is produced by podocytes and not endothelial cells.

The objective of this study was to combine a successful strategy for treatment of Alport syndrome with a safe and successful gene delivery method such that COL4A3, COL4A4 or COL4A5 gene expression could be delivered to podocytes, preferably at an early stage of disease and before the onset of proteinuria.

Example 1 design, construction and testing of minimal nephrin promoters coupled to COL4A3, COL4A4 and COL4A5

Design and construction of AAV constructs

The following AAV transfer plasmids comprising COL4A3, COL4A4 and COL4A5 coupled to the mini nephrin promoter were designed and constructed ("265" -detailed information on design, construction and testing of mini nephrin promoter, see example 2):

paav.265.COL4A3.3flag. Sv40, AAV plasmid, comprising COL4A3 coupled to the mini nephrin promoter (see fig. 4A).

pAAV.265.Col4a4.3flag.sv40, AAV plasmid, comprising COL4A4 coupled to the mini nephrin promoter (see FIG. 4B)

pAAV.265.Col4a5.3flag. Sv40, AAV plasmid, comprising COL4A5 coupled to the mini nephrin promoter (see FIG. 4C)

SmaI digestion was performed to confirm the identity of the plasmid (see FIGS. 4D-E). This indicates that COL4a3, a4 and a5 were successfully cloned into AAV having a 265bp mini nephrin promoter and SV40polyA tail.

Testing of AAV constructs

The following AAV viral vectors were prepared using standard methods:

aav.col4a3.nephrin265.Sv40 with LK03 serotype

Aav.col4a5.Nephrin265.Sv40 with LK03 serotype

AAV.COL4A5.Nephrin265.Sv40 with serotype 2/9

Figure 5A shows immunoprecipitation experiments of full-length FLAG-tagged Col4a3 (LK 03) or Col4a5 (LK 03) in human differentiated ciPodocyte pulled with anti-FLAG antibody. The anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human FLAGIgG was used as a control.

Fig. 5B shows a western blot of protein lysates showing the expression levels of Col4a3 (LK 03 capsid serotype), col4a5 (LK 03), and Col4a5 (2/9 capsid serotype) in human or mouse differentiated cipodocytes. Cipodocytes from uninfected humans and mice were used as controls.

FIG. 5C shows confocal images showing immunofluorescence staining of transduced Col4a5 and F-actin in human wild-type CiPodocyte/Col4a53 xFlagaAAVCiPodocyte. Col4a5 was present at cytosolic levels in human differentiated podocytes infected with Col4a53xFlagAAV virus compared to the wild type counterpart.

These results indicate that, surprisingly, we successfully transduced human podocytes with full-length COL4a3 and COL4a5 coupled to the mini nephrin promoter, and that the mini nephrin promoter surprisingly drives expression of full-length COL4a3 or COL4a5 in human podocytes.

Example 2 design, construction and testing of the minimal nephrin promoter

Design of the minimal nephrin promoter

The human NPHS1 promoter has been described in Moeller et al 2002j Am Soc Nephrol,13 (6): 1561-7 and Wong MA et al.2000Am J Physiol Renal Physiol,279 (6): f1027-32. This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific. This is hereinafter referred to as the "FL" nephrin promoter and is shown in FIG. 6A.

The FL nephrin promoter was initially cleaved to 822bp (819 bp, excluding the start codon) by deletion of the N-terminal sequence. This is hereinafter referred to as the "midi" nephrin promoter and is shown in figure 6B.

The midi nephrin promoter was further cleaved to 268bp (265 bp, excluding the start codon) by removing the putative general transcriptional domain from the central region. This is hereinafter referred to as the "mini" nephrin promoter and is shown in figure 6C.

Construction of vector constructs

Midi nephrin promoter

The pACE _ hNPHS1 promoter was used as a template to introduce BamHI and ClaI restriction sites as shown in FIG. 7A. The fragments were then gel extracted and digested with ClaI and BamHI at 37 ℃ for 1 hour before ligation into pLenti GFP Blast vector. The ligation was further transformed into stable competent E.coli (E.coli) cells, DNA extracted and sequenced (Midi promoter). The final lentiviral vector is shown in figure 7B.

Mini nephrin promoter

The pACE _ hNPHS1 promoter was used to perform PCR on the Overhang (OH), as shown in FIG. 8A. Two portions of the OH-containing promoter were gel extracted for NEBuilder HiFi Assembly reaction on the pLenti GFP Blast vector. The ligation reaction was then cleaned using a DNA clean-up kit and then transformed into stable competent e. DNA was extracted and sequenced. The final lentiviral vector is shown in figure 8B.

Test vector constructs

GFP was expressed in an in vitro cell model using a minimal nephrin promoter to examine efficacy and podocyte specificity.

The pLenti GFP BlastNephrin promoter construct (full length, midi and Mini) was used to transfect HEK293T cells for 48 hours to prepare viruses that were further used to create human conditionally immortalized podocytes stably expressing GFP-tagged FLNPHS1, midi or Mini promoters.

Conditionally immortalized human podocytes (cipodocytes) were transfected with lentiviral vectors to determine if the minimal promoter was able to drive GFP expression. Both midi and mini nephrin promoters were shown to drive GFP expression. Fig. 9 shows representative fluorescence microscopy images showing GFP expression from the mini nephrin promoter. Figure 10 shows a representative western blot showing GFP expression from the mini nephrin promoter. These results indicate that the minimal nephrin promoter is capable of driving transgene expression in podocytes.

Lentiviral vectors are also used to transduce human glomerular cells. ciPodocyte and glomerular endothelial cells were transduced with a lentivirus containing GFP coupled to a minimal nephrin reporter. FIGS. 11A-C show FACS analysis demonstrating median GFP fluorescence (AFU) for all viable single peaks of conditionally-immortalized human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte analyzer. Untransduced cells (cell control) were compared to cells transduced with lentiviral constructs carrying a GFP expression cassette controlled by either the full-length human nephrin promoter (hnph 1. GFP) or the mini human nephrin promoter (265. GFP). All cells were differentiated for 10 days, trypsinized (100 uL) and diluted in PBS, 2% FBS, 1: 1000 DRAQ7 (150 uL). Data and error bars represent 3 technical replicates (100 uL, > 2500 cells). + -. SEM. These results show podocyte specificity of the minimal nephrin promoter when compared to glomerular endothelial cells.

Example 3 podocyte-targeted Gene therapy

We have developed targeted gene delivery systems in human and mouse podocytes using adeno-associated virus (AAV) (see PCT/GB 2020/050097). AAV serotype 2/9 successfully infects podocytes in vivo using podocyte-specific promoter (nephrin), inducing podocin expression. AAV treatment successfully restored podocin expression and improved proteinuria in animals that caused proteinuria by knockdown of podocin using the Cre-Loxp system (NPHS 2 fl/fl). Furthermore, we have shown efficient and specific transduction of GFP by AAV LK03 (more efficient than AAV 2/9) in human podocytes using the same promoter.

Reference to the literature

KODIPPILI K，HAKIM CH，PAN X，YANG HT，YUE Y，ZHANG Y，SHIN JH，YANG NN，DUAN D.Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model.Hum Gene Ther.2018 Mar；29(3)：299-311.

LUO，X.，HALL，G.，LI，S.，BIRD，A.，LAVIN，P.J.，WINN，M.P.，KEMPER，A.R.，BROWN，T.T.&KOEBERL，D.D.2011.Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.Mol Ther，19，1961-70.

MCCLEMENTS ME，MACLAREN RE.Adeno-associated Virus(AAV)Dual Vector Strategies for Gene Therapy Encoding Large Transgenes.Yale J Biol Med.2017 Dec 19；90(4)：611-623

MOELLER，M.J.，SANDEN，S.K.，SOOFI，A.，WIGGINS，R.C.&HOLZMAN，L.B.2002.Two gene fragments that direct podocyte-specific expression in transgenic mice.J Am Soc Nephrol，13，1561-7.

PICCONI，J.L.，MUFF-LUETT，M.A.，WU，D.，BUNCHMAN，E.，SCHAEFER，F.&BROPHY，P.D.2014.Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9.Molecular Therapy.Methods&Clinical Development，1，14014.

ROCCA，C.J.，UR，S.N.，HARRISON，F.&CHERQUI，S.2014.rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery：conclusion of a comparative study.Gene therapy，21，618-628.

SCHAMBACH，A.，BOHNE，J.，BAUM，C.，HERMANN，F.G.，EGERER，L.，VON LAER，D.&GIROGLOU，T.2005.Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression.Gene Therapy，13，641.SCHIEVENBUSCH，S.，STRACK，I.，SCHEFFLER，M.，NISCHT，R.，COUTELLE，O.，

M.，HALLEK，M.，FRIES，J.W.U.，DIENES，H.-P.，ODENTHAL，M.&

H.2010.Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis.Molecular Therapy，18，1302-1309.

Detailed description of the preferred embodiments

Various features and embodiments of the invention will now be described with reference to the following numbered paragraphs (para).

1. A viral vector gene therapy, wherein the viral vector comprises:

COL4A3, COL4A4 or COL4A5 transgenes; and

optionally a podocyte-specific promoter.

2. The viral vector gene therapy according to paragraph 1, wherein the podocyte-specific promoter is the minimum nephrin promoter NPHS1 or podocin promoter NPHS2.

3. The viral vector gene therapy according to

paragraph

1 or 2, wherein the viral vector is an adeno-associated virus (AAV).

4. A viral vector gene therapy according to paragraph 3, wherein the AAV vector is AAV serotype 2/9, LK03 or 3B.

5. The viral vector gene therapy according to any of paragraphs 1 to 4, wherein the COL4A3, COL4A4 or COL4A5 transgene is a minigene.

6. The viral vector gene therapy according to any one of paragraphs 1 to 4, wherein the gene therapy comprises:

optionally a podocyte-specific promoter; and

a second viral vector comprising at least a portion of a respective COL4A3, COL4A4, or COL4A5 transgene; and

optionally a podocyte-specific promoter.

7. A viral vector gene therapy according to any one of paragraphs 1 to 6, wherein the viral vector further comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

8. A viral vector gene therapy according to any of paragraphs 1 to 7, wherein the COL4A3, COL4A4 or COL4A5 transgene is human and/or comprises a Hemagglutinin (HA) tag.

9. The viral vector gene therapy according to any of paragraphs 1 to 8, wherein the viral vector further comprises a Kozak sequence between the promoter and the COL4A3, COL4A4 or COL4A5 transgene.

10. The viral vector gene therapy according to any of paragraphs 1 to 9, wherein the viral vector further comprises a polyadenylation signal, such as the bovine growth hormone (bGH) polyadenylation signal.

11. A viral vector gene therapy according to any one of paragraphs 1 to 10 for use in the treatment or prevention of Alport syndrome.

12. The viral vector gene therapy for use according to paragraph 11, wherein the viral vector gene therapy is to be administered to a human patient.

13. The viral vector gene therapy for use according to paragraphs 11 or 12, wherein the viral vector gene therapy is administered systemically.

14. The viral vector gene therapy for use according to any one of paragraphs 11 to 13, wherein the viral vector gene therapy is to be administered by intravenous injection.

15. The viral vector gene therapy for use according to any one of paragraphs 11 to 14, wherein the viral vector gene therapy is to be administered by injection into the renal artery.

Claims

1. A viral vector, wherein said viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

2. A viral vector according to claim 1, wherein:

(i) The COL4A3 transgene encodes a COL4A3 polypeptide or fragment thereof, the COL4A3 polypeptide comprising a sequence identical to SEQ ID NO:1, or a polypeptide sequence having at least 70% identity to SEQ ID NO:1, a polypeptide sequence having at least 70% identity;

(ii) The COL4A4 transgene encodes a COL4A4 polypeptide or fragment thereof, the COL4A4 polypeptide comprising a sequence identical to SEQ ID NO:2, or a polypeptide sequence having at least 70% identity to SEQ ID NO:2 a polypeptide sequence having at least 70% identity; and/or

(iii) The COL4A5 transgene encodes a COL4A5 polypeptide or fragment thereof, the COL4A5 polypeptide comprising a sequence identical to SEQ ID NO:3, or a polypeptide sequence having at least 70% identity to SEQ ID NO:3 with at least 70% identity.

3. A viral vector according to claim 1 or 2, wherein: (i) The COL4A3 transgene encodes a full-length COL4A3 polypeptide, (ii) the COL4A4 transgene encodes a full-length COL4A4 polypeptide; and/or (iii) the COL4A5 transgene encodes a full-length COL4A5 polypeptide.

4. The viral vector according to any one of the preceding claims, wherein the viral vector comprises a podocyte-specific promoter.

5. The viral vector according to any one of the preceding claims, wherein said podocyte-specific promoter is the minimum nephrin promoter NPHS1 or podocin promoter NPHS2, preferably wherein said podocyte-specific promoter is the minimum nephrin promoter NPHS1.

6. The viral vector according to claim 5, wherein said minimal nephrin promoter NPHS1 comprises the sequence as set forth in SEQ ID NO:10 or a nucleotide sequence corresponding to SEQ ID NO:10, or a variant consisting of a sequence as set forth in SEQ ID NO:10 or a nucleotide sequence corresponding to SEQ ID NO:10 variant compositions that are at least 70% identical.

7. A viral vector according to any one of the preceding claims, wherein the viral vector is an adeno-associated virus (AAV).

8. A viral vector according to claim 7, wherein the AAV vector is in the form of an AAV vector particle.

9. A viral vector according to claim 7 or 8, wherein said AAV vector particle is a podocyte-specific AAV vector.

10. A viral vector according to any one of claims 7 to 9, wherein said AAV vector is AAV serotype 2/9, LK03 or 3B.

11. A viral vector according to any of the preceding claims, wherein the COL4A3, COL4A4 or COL4A5 transgene is a minigene.

12. The viral vector according to any one of the preceding claims, wherein the viral vector further comprises a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

13. The viral vector according to any one of claims 1 to 11, wherein the viral vector does not comprise a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

14. The viral vector according to any of the preceding claims, wherein the COL4A3, COL4A4 or COL4A5 transgene is human and/or comprises a Hemagglutinin (HA) tag.

15. A viral vector according to any of the preceding claims, wherein said viral vector further comprises a Kozak sequence between said promoter and said COL4A3, COL4A4 or COL4A5 transgene.

16. The viral vector according to any one of the preceding claims, wherein the viral vector additionally comprises a polyadenylation signal, such as the bovine growth hormone (bGH) polyadenylation signal or the early SV40 polyadenylation signal.

17. A viral vector according to claim 16, wherein said polyadenylation signal is the early SV40 polyadenylation signal.

18. Viral vector gene therapy, wherein the gene therapy comprises:

19. Viral vector gene therapy according to claim 18, wherein the first viral vector is a viral vector as defined in any one of claims 1 to 17 and/or the second viral vector is a viral vector as defined in any one of claims 1 to 17.

20. A viral vector or viral vector gene therapy according to any one of the preceding claims for use in the treatment or prevention of Alport syndrome.

21. A viral vector or viral vector gene therapy for use according to claim 20, wherein said viral vector or viral vector gene therapy is administered to a human patient.

22. A viral vector or viral vector gene therapy for use according to claim 20 or 21, wherein said viral vector or viral vector gene therapy is administered systemically.

23. A viral vector or viral vector gene therapy for use according to any one of claims 20 to 22, wherein the viral vector or viral vector gene therapy is administered by intravenous injection.

24. A viral vector or viral vector gene therapy for use according to any one of claims 20 to 23, wherein the viral vector or viral vector gene therapy is administered by injection into the renal artery.